The efficient production of bioactive proteins and peptides has become a hallmark of the biomedical and industrial biochemical industry. Bioactive peptides and proteins are used as curative agents in a variety of diseases such as diabetes (insulin), viral infections and leukemia (interferon), diseases of the immune system (interleukins), and red blood cell deficiencies (erythropoietin) to name a few. Additionally, large quantities of proteins and peptides are needed for various industrial applications including, for example, the pulp and paper and pulp industries, textiles, food industries, personal care and cosmetics industries, sugar refining, wastewater treatment, production of alcoholic beverages and as catalysts for the generation of new pharmaceuticals.
With the advent of the discovery and implementation of combinatorial peptide screening technologies such as bacterial display, yeast display, phage display, ribosome display, and mRNA display technology new applications for peptides having strong affinity for a target surface have been developed. In particular, peptides are being looked to as linkers in biomedical fields for the attachment of diagnostic and pharmaceutical agents to surfaces (see Grinstaff et al, U.S. Patent Application Publication No. 2003-0185870 and Linter in U.S. Pat. No. 6,620,419), as well as in the personal care industry for the attachment of benefit agents to body surfaces such as hair and skin (see commonly owned U.S. Pat. No. 7,220,405, and Janssen et al. U.S. Pat. No. 7,129,326), and in the printing industry for the attachment of pigments to print media (see commonly owned U.S. Patent Application Publication No. 2005-0054752).
In some cases commercially useful proteins and peptides may be synthetically generated or isolated from natural sources. However, these methods are often expensive, time consuming and characterized by limited production capacity. The preferred method of protein and peptide production is through the fermentation of recombinantly constructed organisms, engineered to over-express the protein or peptide of interest. Although preferable to synthesis or isolation, recombinant expression of peptides has a number of obstacles to be overcome in order to be a cost-effective means of production. For example, peptides (and in particular short peptides) produced in a cellular environment are susceptible to degradation from the action of native cellular proteases. Additionally, purification can be difficult, resulting in poor yields depending on the nature of the protein or peptide of interest.
One means to mitigate the above difficulties is the use of genetic chimera for protein and peptide expression. A chimeric protein or “fusion protein” is a polypeptide comprising at least one portion of a desired protein product fused to at least one portion comprising a peptide tag. The peptide tag may be used to assist protein folding, assist in purification, alter polypeptide solubility, protect the protein from the action of degradative enzymes, and/or assist the protein in various transport and targeting processes.
In many cases it is useful to express a protein or peptide in insoluble form, particularly when the peptide of interest is rather short, normally soluble, and/or subject to proteolytic degradation within the host cell. Production of the peptide in insoluble form both facilitates simple recovery and protects the peptide from undesirable proteolytic degradation. One means to produce the peptide in insoluble form is to recombinantly produce the peptide as part of an insoluble fusion protein by including in the fusion construct, at least one peptide tag (i.e., an inclusion body tag) that induces inclusion body formation. Typically, the fusion protein is designed to include at least one cleavable peptide linker so that the peptide of interest can be subsequently recovered from the fusion protein. The fusion protein may be designed to include a plurality of solubility tags, cleavable peptide linkers, and regions encoding the peptide of interest.
Fusion proteins comprising a peptide tag that facilitate the expression of insoluble proteins are well known in the art. Typically, the tag portion of the chimeric or fusion protein is large, increasing the likelihood that the fusion protein will be insoluble. Examples of large peptides that are typically used include, but are not limited to chloramphenicol acetyltransferase (Dykes et al., Eur. J. Biochem., 174:411 (1988), β-galactosidase (Schellenberger et al., Int. J. Peptide Protein Res., 41:326 (1993); Shen et al., Proc. Nat. Acad. Sci. USA 281:4627 (1984); and Kempe et al., Gene, 39:239 (1985)), glutathione-S-transferase (Ray et al., Bio/Technology, 11:64 (1993) and Hancock et al. (WO94/04688)), the N-terminus of L-ribulokinase (U.S. Pat. No. 5,206,154 and Lai et al., Antimicrob. Agents & Chemo., 37:1614 (1993), bacteriophage T4 gp55 protein (Gramm et al., Bio/Technology, 12:1017 (1994), bacterial ketosteroid isomerase protein (Kuliopulos et al., J. Am. Chem. Soc. 116:4599 (1994), ubiquitin (Pilon et al., Biotechnol. Prog., 13:374-79 (1997), bovine prochymosin (Naught et al., Biotechnol. Bioengineer. 57:55-61 (1998), and bactericidal/permeability-increasing protein (“BPI”; Better, M. D. and Gavit, P D., U.S. Pat. No. 6,242,219). The art is replete with specific examples of this technology, see for example U.S. Pat. No. 6,613,548, describing fusion protein of proteinaceous tag and a soluble protein and subsequent purification from cell lysate; U.S. Pat. No. 6,037,145, teaching a tag that protects the expressed chimeric protein from a specific protease; U.S. Pat. No. 5,648,244, teaching the synthesis of a fusion protein having a tag and a cleavable linker for facile purification of the desired protein; and U.S. Pat. No. 5,215,896; U.S. Pat. No. 5,302,526; and U.S. Pat. No. 5,330,902; and U.S. Patent Application Publication No. 2005-221444, describing fusion tags containing amino acid compositions specifically designed to increase insolubility of the chimeric protein or peptide.
Shorter solubility tags have been developed from the Zea mays zein protein (co-owned U.S. Pat. No. 7,732,569) the Daucus carota cystatin (co-owned U.S. Pat. No. 7,662,913), and an amyloid-like hypothetical protein from Caenorhabditis elegans (co-owned U.S. Pat. No. 7,427,656; each hereby incorporated by reference in their entirety.) The use of short inclusion body tags increases the yield of the target peptide produced within the recombinant host cell.
Aspartic acid-proline linkages can be cleaved using acid treatment. However, the conditions typically used include at least one strong acid, such as HCl or H2SO4, and may require subsequent neutralization with base and may increase the cost of peptide recovery due the amount of salt produced. Further, acid hydrolysis conditions for the intended aspartic acid-proline pair may be accompanied by undesirable hydrolysis at other sites where aspartic acid residues occur or may lead to the deamidation of glutamine or asparagine.
One problem to be solved is to provide peptide linkers that are more sensitive to acid hydrolysis when compared to a single aspartic acid-proline linkage. Increased sensitivity may permit the use of weaker acids, reduce the amount of base that may be needed for neutralization, and may help to protect the peptide of interest from unwanted hydrolysis at other locations within the peptide of interest.
Situations may occur where a peptide or protein of interest contains one or more acid labile aspartic acid-proline linkages where acid hydrolysis is not desired. As such, another problem to be solved is to provide a method to increase the stability of aspartic acid-proline linkages to acid treatment in peptides or proteins wherein acid hydrolysis is undesirable.